Linus Pauling Institute

Annie L. Cao, Biological and Population Health Sciences, Oregon State University
Laura M. Beaver, Biological and Population Health Sciences, Linus Pauling Institute, Oregon State University
Emily Ho, Biological and Population Health Sciences, Moore Family Center for Whole Grain Foods, Linus Pauling Institute, Oregon State University

Insulin is a vital hormone secreted by beta cells in the pancreas to lower glucose levels in the blood. Zinc is an essential micronutrient that plays an important role in insulin storage, signaling, and secretion. Arsenic is an environmental toxicant that millions of people are chronically exposed to, and this exposure can co-exist with zinc deficiency. Zinc deficiency and inorganic arsenic exposure have both been independently associated with an increased risk of developing diabetes, but it is not known whether they interact to promote diabetes. Here, we tested the hypothesis that arsenic can deplete zinc levels in pancreatic beta cells, and co-exposure to arsenic and zinc deficiency alters insulin regulation and production. Rat pancreatic beta cells were cultured in zinc adequate or zinc deficient media and exposed to arsenic at 0, 50, or 500 ppb concentrations. The combination of zinc deficiency and arsenic exposure decreased cell proliferation and increased cell death more than either zinc deficiency or arsenic exposure alone. Arsenic dose-dependently altered the expression of the transcription factors (Neurod1, Pdx1) that regulate insulin production at the mRNA level, while zinc deficiency did not significantly affect their expression. There were no significant effects of zinc deficiency, or arsenic exposure, on the mRNA levels of the pancreatic-specific zinc transporter Znt8. Arsenic significantly downregulated Glut2 mRNA levels at the 500 ppb dose. Arsenic also decreased insulin levels secreted by the beta cells, and suppressed the expression of the gene that produces insulin (Ins1) at the 500 ppb dose. In contrast, zinc deficiency upregulated expression of Ins1, and increased insulin release. This project has enhanced the understanding of the relationships between zinc, arsenic, and insulin and further investigation of this topic can influence the prevention and/or treatment of diabetes.

Laura M. Beaver, Biological and Population Health Sciences, Linus Pauling Institute, Oregon State University
Lisa Truong, Department of Environmental and Molecular Toxicology, Oregon State University
Carrie L. Barton, Department of Environmental and Molecular Toxicology, Oregon State University
Tyler T. Chase, Biological and Population Health Sciences, Oregon State University
Greg D. Gonnerman, Department of Environmental and Molecular Toxicology, Oregon State University
Carmen P. Wong, Linus Pauling Institute, Biological and Population Health Sciences, Oregon State University
Robert L. Tanguay, Department of Environmental and Molecular Toxicology, Oregon State University
Emily Ho, Biological and Population Health Sciences, Moore Family Center for Whole Grain Foods, Linus Pauling Institute, Oregon State University

Zinc deficiency and chronic low level exposures to inorganic arsenic in drinking water are both significant public health concerns that affect millions of people including pregnant women. These two conditions often co-exist in the human population, but little is known about their interaction, and in particular whether zinc deficiency sensitizes individuals to arsenic exposure and toxicity, especially during critical windows of development. To address this, we utilized the Danio rerio (zebrafish) model to test the hypothesis that parental zinc deficiency sensitizes the developing embryo to low-concentration arsenic toxicity, leading to altered developmental outcomes. Adult zebrafish were fed a defined diet that contained either 33.8 ug/ g zinc (zinc adequate) or 14.5 ug/g zinc (zinc deficient) and subsequently spawned to produce zinc adequate or zinc deficient embryos respectively. The zinc adequate and zinc deficient embryos were then treated with environmentally relevant concentrations of 0, 50, and 500 ppb arsenic. Arsenic exposure significantly reduced the amount of zinc in the developing embryo by ~7%. The combination of zinc deficiency and low-level arsenic exposures did not sensitize the developing embryo to increased developmental malformations or mortality. The combination did cause a 40% decline in physical activity of the embryos, and this decline was significantly greater than what was observed with zinc deficiency or arsenic exposure alone. Significant changes in RNA expression of genes that regulate zinc homeostasis, response to oxidative stress and insulin production (including zip1, znt7, nrf2, ogg1, pax4, and insa) were found in zinc deficient, or zinc deficiency and arsenic exposed embryos. Overall the data suggests that the combination of zinc deficiency and arsenic exposure has harmful effects on the developing embryo and may increase the risk for developing chronic diseases like diabetes.

Carmen P. Wong, Biological and Population Health Sciences, Linus Pauling Institute, Oregon State University
Tyler Chase, Biological and Population Health Sciences, Oregon State University
Erica Dashner, University of New Mexico Health Sciences Center Laurie Hudson, University of New Mexico Health Sciences Center
Emily Ho, Biological and Population Health Sciences, Moore Family Center for Whole Grain Foods, Linus Pauling Institute, Oregon State University

Zinc is an essential micronutrient important in many biological processes. Zinc has antioxidant and anti-inflammatory properties, and zinc deficiency results in increased oxidative stress and inflammatory response. It is estimated that 12% of Americans do not consume adequate zinc, and are at risk for marginal zinc deficiency. Arsenic is a naturally occurring element found in the environment. Arsenic contamination is a major public health concern in the United States and worldwide. Arsenic exposure shares many hallmarks of zinc deficiency, including increased inflammation and oxidative stress, and both can contribute to the development of chronic diseases including cardiovascular disease, diabetes, and cancer. Notably, populations at risk for arsenic exposure also have co-existing signs of zinc deficiency. To date, very little is known regarding the interactions between zinc status and arsenic exposure, and whether zinc status impacts the susceptibility of arsenic toxicity and vice versa. The goal of this study was to examine the combined effects of zinc deficiency and arsenic exposure on oxidative stress and inflammatory response. Our hypothesis was that zinc deficiency sensitizes cells to arsenic-induced toxicity by amplifying oxidative stress and inflammatory response. In cell culture, zinc deficient THP1 monocytes had reduced intracellular zinc and increased proinflammatory (IL8) and oxidative stress (HMOX-1) response. Exposure to arsenic (0.1-10uM) resulted in further reduction in intracellular zinc, and enhanced IL8 and HMOX-1 expression. In animal models, marginally zinc deficient mice had further decrease in zinc status when exposed to arsenic (50-500ppb) in the drinking water. Zinc deficient mice had increased baseline expression of inflammatory markers in the liver. Upon LPS challenge to elicit an acute inflammatory response, arsenic-exposed mice had a further increase in proinflammatory response. Our data suggest that arsenic exposure interacts with zinc deficiency to further reduce overall zinc status, and amplified oxidative stress and proinflammatory response.

Shilpy Dixit, Graduate Program in Neuroscience, Vanderbilt University
Joshua P. Fessel, Vanderbilt University Medical Center
Fiona E. Harrison, Vanderbilt University Medical Center

Mitochondrial dysfunction is elevated in very early stages of Alzheimer’s disease and exacerbates oxidative stress, which contributes to disease pathology. We show that ascorbate is critical in supporting mitochondrial function, including oxygen consumption and energy production. Mitochondria were isolated from 4-month-old wild-type mice, transgenic mice carrying the APPSWE and PSEN1dE9 mutations, mice with decreased brain and mitochondrial ascorbate (vitamin C) via heterozygous knockout of the sodium dependent vitamin C transporter, type 2 (SVCT2+/-) and the combined mutant (SVCT2+/-;APP/PSEN1). In closed high-resolution respirometry chambers, mitochondrial isolates from SVCT2+/- mice consumed less oxygen and exhibited decreased mitochondrial membrane potential compared to wild-type isolates. Conversely, isolates from APP/PSEN1 mice consumed more oxygen, and exhibited an increase in mitochondrial membrane potential, but had a significantly lower ATP/ADP ratio compared to wild type isolates. Greater levels of reactive oxygen species were produced in mitochondria isolated from both APP/PSEN1 and SVCT2+/- mice compared to wild-type isolates. Acute administration of ascorbate to isolated wild-type mitochondria increased oxygen consumption compared with untreated mitochondria suggesting ascorbate may support energy production. Additionally, mitochondria isolated from mice with SVCT2 over-expression (SVCT2Tg) show a decrease in reactive oxygen species with no change in mitochondrial function compared to wild type isolates. This study provides further evidence that mitochondrial dysfunction can occur at an early, prodromal stage of Alzheimer’s disease, prior to the presence of amyloid β aggregates and cognitive deficits, and that ascorbate deficiency leaves mitochondria vulnerable to oxidative damage, thereby contributing to Alzheimer’s disease progression. Adequate ascorbate intake is an effective and inexpensive mitochondrial-targeted antioxidant that supports brain health. It can provide a potent preventative strategy against neurodegenerative disease, particularly in the populations most at risk for Alzheimer’s disease in which stores are often depleted through mitochondrial dysfunction and elevated oxidative stress.

Yang Zhang, School of Public Health and Human Sciences, Linus Pauling Institute, Oregon State University
Hongjun Wang, Department of Surgery-Transplant and Mary & Dick Holland Regenerative Medicine Program, University of Nebraska Medical Center
Mai Le, Department of Biochemistry and Biophysics, College of Pharmacy, Oregon State University
Arup Indra, Department of Biochemistry and Biophysics, College of Pharmacy, Linus Pauling Institute, Oregon State University, Department of Dermatology, Knight Cancer Institute, Oregon Health & Science University
Gitali Indra, College of Pharmacy, Oregon State University, Knight Cancer Institute, Oregon Health & Science University
Jingwei Xie, Department of Surgery-Transplant and Mary & Dick Holland Regenerative Medicine Program, University of Nebraska Medical Center
Adrian F. Gombart, Department of Biochemistry and Biophysics, Linus Pauling Institute, Oregon State University

Multi-drug resistant bacteria are a major challenge for treatment of surgical site and traumatic wound infections. Human cathelicidin antimicrobial peptide (CAMP) is critical for skin barrier defense, wound healing and can effectively combat drug resistant bacterial infections. Recently, we demonstrated that 25(OH)D3 encapsulated in poly(e-caprolactone) (PCL) nanofibers was released in a sustained manner and induced CAMP gene expression in keratinocyte and monocyte cell lines for up to five days in vitro. We propose using this novel strategy to achieve effective and constant local delivery of 1α,25(OH)2D3 to enhance wound healing and minimize the potential risk of infections by augmenting innate immune responses. Treatment of human myeloid leukemia cell line U937 with 1α,25(OH)2D3 loaded PCL (PCL-D) fibers significantly induced sustained cathelicidin mRNA and protein expression for five days. In vivo studies with a human CAMP transgenic mouse model showed a statistically significant 1.5-fold increase in human CAMP mRNA expression after three days in the wounds containing PCL-D fibers versus PCL only. Furthermore, increased expression of Cyp24a1 was observed in the kidney of PCL-D treated animals indicating systemic release of 1α,25(OH)2D3 from the PCL nanofibers. Also, PCL-D induced CAMP protein expression in human skin explants in vitro. In conclusion, 1α,25(OH)2D3 loaded PCL nanofiber dressings significantly induced both CAMP mRNA and protein levels in cell lines, a transgenic mouse model and in human skin. These preliminary findings suggest that we could enhance innate immunity by inducing antimicrobial peptide production and possibly mitigate the selection for multidrug resistance and improve wound healing. Future work will focus on developing these biocompatible and biodegradable wound dressings for this purpose using the model systems described above. Furthermore, we will identify the molecular mechanisms by which vitamin D improves barrier defenses and enhances wound healing.

Melissa McDougall, College of Public Health and Human Sciences, Linus Pauling Institute, Oregon State University
Scott W. Leonard, Linus Pauling Institute, Oregon State University
Jaewoo Choi, Linus Pauling Institute, Oregon State University
Kathy R. Magnusson, College of Veterinary Medicine, Linus Pauling Institute, Oregon State University
Lisa Truong, Environmental and Molecular Toxicology, Sinnhuber Aquatic Research Laboratory, Oregon State University
Robert Tanguay, Environmental and Molecular Toxicology, Sinnhuber Aquatic Research Laboratory, Linus Pauling Institute, Oregon State University
Maret G. Traber, College of Public Health and Human Sciences, Linus Pauling Institute, Oregon State University

Zebrafish (Danio rerio) are a recognized model for studying consequences of increased oxidative stress within the brain. The lipophilic antioxidant vitamin E (α-tocopherol; VitE) has an established role in neurological health and cognitive function, but the mechanisms involved remain unknown. We hypothesized that VitE deficient zebrafish would display cognitive impairments associated with elevated lipid peroxidation and metabolic disruptions in the brain. In the present study, we investigated behavioral perturbations due to chronic VitE deficiency in adult zebrafish fed from 45 days to 18-months of age diets that were either VitE-deficient (E–) or VitE-sufficient (E+). Quantified α-tocopherol concentrations at 18-months in E– brains (5.7 ± 0.1 nmol/g tissue) were ~20-times lower than in E+ (122.8 ± 1.1; n= 10/group). We found that chronic VitE deficiency impairs both associative (avoidance conditioning) and non-associative (habituation) learning in adult zebrafish. Further, inadequate brain VitE led to altered brain PL and lysoPL compositions, and perturbed choline and methyl-donor metabolites. The depletion of choline and the dysregulation of PC composition suggests that methyl donor availability became limiting as the animal attempted to correct the loss of phosphatidyl choline with docosahexanoic acid (DHA-PC). VitE deficiency also resulted in decreases in brain metabolite concentrations in the glycolytic pathway, the pentose phosphate pathway and the citric acid cycle; these outcomes suggest that the energy demand increased in order to repair the damage caused by increased lipid peroxidation and thus altered steady-state energy metabolism. Increases in ß-hydroxybutyrate and ketogenic substrates further emphasize that the ketone availability is increased in the E– brain. Taken together, our findings suggest that VitE plays a major role in preventing the dysregulation of brain energy metabolism by protecting lipids from increased peroxidation, and that its deficiency induces a variety of metabolic alterations that have largely been under appreciated. Although VitE deficiency leads to major alterations in lipid and energy metabolites, apparently resulting from metabolic responses that allow the animal to function in the face of increased lipid peroxidation and oxidative damage, these changes are insufficient to prevent major deficits in cognitive function in the aging fish.

Thomas E. Levy, PeakEnergy.com

Vitamin C has been documented in the scientific literature to be a powerful therapy for resolving and helping to resolve a wide variety of infections, often as a monotherapy. Viral infections have been proven to be especially susceptible. As vitamin C has been shown to support and stimulate immune function by many different mechanisms, clinical research is now clearly showing that it can be expected to augment, sometimes additively and often synergistically, multiple other effective traditional and non-traditional protocols for the treatment of infectious diseases. To date, no viruses have been reported that are not effectively treated/resolved by vitamin C, whether in the test tube, tissue preparations, the laboratory animal, or the human. This includes Dengue, Chikungunya, Zika, and Ebola infections. It now appears that using sufficient doses of different forms of vitamin C, such as oral liposome-encapsulated forms, can achieve comparable results to the more traditional intravenous forms of administration. The addition of ozone, hydrocortisone, zinc, antibiotics, and other anti-infection measures to vitamin C all appear to have often profound clinical impacts on infections still felt to be effectively untreatable by traditional medicine. The Multi-C Protocol will also be briefly discussed, as the high dosing of multiple forms of vitamin C appears to sometimes have effects not achievable with one form alone.